ABSTRACT
The synthesis of a variety of N-alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles is reported herein. Their potential as antifungal agents is evaluated by preliminary screening against Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), and Candida albicans (C. albicans). Statistical analyses illustrate a strong relationship between chain length and growth inhibition for S. cerevisiae and S. pombe (p < 0.0001 in every case). Of particular interest is the activity of both sets of compounds against S. cerevisiae, as this is emerging as an opportunistic pathogen, especially in immunosuppressed and immunocompromised patients. Bioassays were set up to compare the efficacy of our range of N-alkylated compounds against classic antifungal agents; Amphotericin B and Thiabendazole.
Subject(s)
Antifungal Agents/pharmacology , Benzothiazoles/pharmacology , Candida albicans/drug effects , Indoles/pharmacology , Saccharomyces cerevisiae/drug effects , Schizosaccharomyces/drug effects , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Benzothiazoles/chemical synthesis , Benzothiazoles/chemistry , Candida albicans/growth & development , Dose-Response Relationship, Drug , Indoles/chemical synthesis , Indoles/chemistry , Microbial Sensitivity Tests , Molecular Structure , Saccharomyces cerevisiae/growth & development , Schizosaccharomyces/growth & development , Structure-Activity RelationshipABSTRACT
The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection--especially in live cells. To surmount such limitations, we engineered a plasmid--pIN-G--encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives.
Subject(s)
Genes, Reporter , Membrane Proteins/metabolism , Signal Transduction , Cell Line , Flow Cytometry , Fluorescence , Fluorescent Antibody Technique , Humans , Membrane Proteins/geneticsABSTRACT
In mammalian central nervous system, neurogenesis occurs in the hippocampus and the subventricular zone (SVZ). We used triple transgenic mouse model of Alzheimer's disease (3 x Tg-AD) harbouring three mutant genes (beta-amyloid precursor, presenilin-1 and tau) and their controls (non-Tg) from 2 to 12 months of age to establish the link between AD and SVZ neurogenesis. We determined the number of SVZ proliferating cells by the presence of phosphorylated histone H3, and their colocalization with glial fibrillary acidic protein to exclude glial phenotype. Less than 2% of histone H3-labelled cells displayed glial fibrillary acidic protein. 3 x Tg-AD mice showed a significant reduction in cell proliferation from 3 months of age that was sustained through all ages, compared with controls. These results indicate that 3 x Tg-AD mice have impaired SVZ cell proliferation, which exacerbates with age.
Subject(s)
Alzheimer Disease/physiopathology , Cell Proliferation , Neurogenesis/physiology , Neurons/physiology , Stem Cells/physiology , Telencephalon/physiopathology , Aging/metabolism , Aging/pathology , Amyloid beta-Protein Precursor/genetics , Animals , Biomarkers/analysis , Biomarkers/metabolism , Disease Models, Animal , Female , Gene Knock-In Techniques , Glial Fibrillary Acidic Protein/analysis , Glial Fibrillary Acidic Protein/metabolism , Histones/analysis , Histones/metabolism , Male , Mice , Mice, Transgenic , Nerve Regeneration/genetics , Neuronal Plasticity/genetics , Neurons/cytology , Phosphorylation , Presenilin-1/genetics , Stem Cells/cytology , Telencephalon/cytology , Transgenes , tau Proteins/geneticsABSTRACT
The endoplasmic reticulum (ER) is the largest intracellular membranous organelle. Functions of the ER are many and diverse, which include various biosynthetic, transport and signalling roles, central to cellular physiology, such as the biosynthesis of membrane and secretory proteins and the regulation of intracellular calcium. Its continuous lumen also serves as a highway for the distribution of proteins and ions to different regions of the cell, independent of the cytosol. The ER is an excitable organelle, capable of generating a regenerative wave of calcium release, which can propagate along the endomembrane throughout the entire cell, serving as a system of intracelluar communication in polarised cells. Nowhere is this feature of ER function more crucial than in neurones. The extremely polarised nature of nerve cells presents a unique challenge for the global co-ordination of localised physiological events such as growth cone guidance and synaptic plasticity. Clearly, the physical continuity of the neuronal ER lumen is central to its functionality as a conduit for communication. To further probe the continuity of ER in neurones and glia, we developed LV-PA-pIN-KDEL, a photoactivatable analogue of our recently described vector LV-pIN-KDEL, a lentivirally delivered ER-targeting soluble GFP. We demonstrate the ability of this vector to transduce astrocytes and neurones in culture and in cortical explants. Furthermore, we exploit the photoactivatable attributes of the vector together with a focal laser photoactivation protocol to reveal the continuous nature of the ER lumen in these cell types, presenting the first direct evidence of an astrocytic ER luminal continuum and providing more data to support the existence of a single ER lumen in neurones.
Subject(s)
Endoplasmic Reticulum/ultrastructure , Lentivirus/genetics , Neuroglia/cytology , Neuroglia/physiology , Neurons/cytology , Neurons/physiology , Transfection/methods , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Humans , Image Enhancement/methods , Kidney/cytology , Kidney/physiology , Light , Microscopy, Fluorescence/methods , Molecular Probe Techniques , RatsABSTRACT
BACKGROUND: The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS: Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION: The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.